Work in model organisms has revealed that a small number of signal transduction pathways, including the Wnt/b-catenin pathway, play key roles throughout development, as well as in tissue repair and stem cell homeostasis in adults. Interestingly, the composition of the Wnt/b-catenin pathway differs in distinct cellular contexts, depending on expression of unique modifiers of the signaling pathway, or expression of different isoforms of conserved pathway components. We and others have evidence (see Preliminary studies) that Wnt/b-catenin signaling is repressed in pluripotent human embryonic stem cells (hESCs) undergoing selfrenewal, and that signaling is active during differentiation into both early and later mesodermal cell lineages. We hypothesize that context-dependent modifiers of Wnt/b-catenin signaling play key roles in the self-renewal and differentiation of hESCs. The initial goal of this proposal is to test the hypothesis that context-dependent modulators of Wnt/b-catenin signaling play key roles in regulating self-renewal and differentiation in hESCs. We believe that pursuit of this goal will reveal detailed mechanisms by which Wnt/beta-catenin signaling regulates self-renewal and specification of cell fate in hESCs. Our second goal is to expand our investigation of the roles of signal transduction pathways in stem cells to include the Hedgehog, Notch, and TGFb pathways, using novel multiplexed fluorescent reporters to enable simultaneous monitoring of multiple pathways in live cells. We believe that the development of the technology to simultaneously visualize the state of activity of multiple signaling pathways in live cells will have numerous uses in studies of signaling pathways in normal and diseased tissues.